Dataset Information

Chromatin immunoprecipitation of ectopic FLAG-tagged TCF7L1 followed by Mass Spectrome-try in H9-TCF7L1 Human Embryonic Stem Cells

ABSTRACT: TCF7L1 is a member of the T cell factor/Lymphoid enhancer factor (TCF/LEF) family of tran-scription factors that are part of the WNT/beta-CATENIN signaling pathway. TCF7L1 modulates transcription by interacting with other regulators on chromatin. TCF7L1 has been shown to be one of the key factors in maintaining pluripotency in human embryonic stem cells (ESCs). We previously demonstrated that the absence of TCF71 causes H9 hESCs to differentiate sponta-neously (Sierra et al., 2018 Development 145(4).pii:dev161075). Mechanistically, how TCF7L1 inhibits differentiation and keeps cells in the pluripotent state is not clear. Identifying transcrip-tional regulators on chromatin is a critical step to elucidating one of several molecular mecha-nisms controlled by TCF7L1. We previously developed a FLAG tagged TCF7L1 transgene that is controlled by a doxycycline-inducible TET-ON system and generated the H9-TCF7L1 line (Sier-ra et al., 2018 Development 145(4).pii:dev161075). Here we used the H9-TCF7L1 line and em-ployed the method called rapid immunoprecipitation (IP) mass spectrometry of endogenous pro-tein (RIME) (Mohammed et al., 2016 Nat Protoc 11(2):316-26) to identify TCF7L1-associated proteins on chromatin. Coupling of these two methods (the FLAG tagged TETON inducible sys-tem and RIME) allowed us to control the level of TCF7L1 expression in hESCs, immunopreci-pate TCF7L1 using an anti-FLAG antibody and capture TCF7L1-bound associated complexes on chromatin. Our MS analysis identified some known proteins that have been shown to associate with the WNT/beta-CATENIN/TCF/LEF pathway, as well as novel complexes that have not been linked with TCF7L1. Gene Ontology analysis suggest these proteins function in chromatin modi-fication, splicing, and RNA processing. Our data could create new ideas for in-depth studies of TCF7L1 controlling pluripotency in human ESCs and for understanding how TCF7L1 may act in other cell types.

INSTRUMENT(S): Orbitrap Fusion, nanoACQUITY UPLC

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Embryonic Stem Cell

SUBMITTER: Linh Vuong

LAB HEAD: Peter J. Donovan

PROVIDER: PXD047582 | Pride | 2024-06-16

REPOSITORIES: Pride

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Publications

Characterization of a chromatin-associated TCF7L1 complex in human embryonic stem cells.

Vuong Linh M LM Pan Songqin S Sierra Robert A RA Waterman Marian L ML Gershon Paul D PD Donovan Peter J PJ

Proteomics 20240417

Human embryonic stem cells (hESCs) resemble the pluripotent epiblast cells found in the early postimplantation human embryo and represent the "primed" state of pluripotency. One factor that helps primed pluripotent cells retain pluripotency and prepare genes for differentiation is the transcription factor TCF7L1, a member of a small family of proteins known as T cell factors/Lymphoid enhancer factors (TCF/LEF) that act as downstream components of the WNT signaling pathway. Transcriptional output ...[more]

PMID: 38629187

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Project description:Embryonic stem cells have potential utility in regenerative medicine due to their pluripotent characteristics. Sall4, a zinc-finger transcription factor, is expressed very early in embryonic development with Oct4 and Nanog, two well characterized pluripotency regulators. Sall4 plays an important role in governing the fate of stem cells through transcriptional regulation of both Oct4 and Nanog. Using chromatin immunoprecipitation coupled to microarray hybridization (ChIP on Chip), we have mapped global gene targets of Sall4 unveiling possible regulation of broad ES cell functions. Approximately 5,000 genes were identified that were bound by the Sall4 protein and many of these have major functions in developmental and regulatory pathways. Sall4 bound more than six times as many annotated genes within promoter regions as Oct4 and twice as many as Nanog. Immunoprecipitation revealed a heterotrimeric protein complex between Sall4, Oct4, and Nanog, consistent with binding site co-occupancies. Further, Sall4 bound many genes that are regulated in part by the chromatin-based epigenetic events mediated by polycomb-repressive complexes and bivalent domains. This suggests that Sall4 plays a central and diverse role in regulating stem cell pluripotency during early embryonic development that involves integration of transcriptional and epigenetic control processes. Keywords: ChIP-chip We used ChIP-chip to map global promoter bound by Sall4, a zinc finger transcription factor. Growing evidence has suggested that Sall4 plays a vital role in ES cell pluripotency maintenance. Evidence for this includes its recent use as a marker for somatic cell reprogramming, in a genetic signature for ES cells, and evidence that Sall4 enhances reprogramming. These recent findings and two previously described interactions with Oct4 and Nanog strongly support the role of Sall4 in ES cells. This hypothesis was furthered here as we show that Sall4 plays important roles through transcriptional regulation of vital ES cell genes.

Dataset Information

Chromatin immunoprecipitation of ectopic FLAG-tagged TCF7L1 followed by Mass Spectrome-try in H9-TCF7L1 Human Embryonic Stem Cells

Publications

Characterization of a chromatin-associated TCF7L1 complex in human embryonic stem cells.

Similar Datasets

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